derivatization-protein-crystals-with-i3c-using-random-microseed

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

The University of Adelaide

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species.

Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici.

Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s ), specificity constant (626 ± 110.0 mm ·s ) and binding free energy change (-37.0 ± 3.5 kcal·mol ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s ) and lowest specificity constant (40.2 ± 4.4 mm ·s ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV).

Nucleoside selectivity of Aspergillus fumigatus nucleoside-diphosphate kinase.

Vanishing white matter (VWM) is a leukodystrophy, caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B)-subunit genes (EIF2B1-EIF2B5); 80% are missense mutations. Clinical severity is highly variable, with a strong, unexplained genotype-phenotype correlation.

Article	Year
Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici. Archives of biochemistry and biophysics\| PubMed ID: 32717225	2020
Nucleoside selectivity of Aspergillus fumigatus nucleoside-diphosphate kinase. The FEBS journal\| PubMed ID: 33089641	2020
Vanishing white matter: Eukaryotic initiation factor 2B model and the impact of missense mutations. Molecular genetics & genomic medicine\| PubMed ID: 33432707	2021

Stephanie Nguyen

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